Alternative export pathways for vector expressed RNA interference

Patent metadata

Field	Value
Publication number	US-10006028-B2
Application number	US-201615165898-A
Country	US
Kind code	B2
Filing date	May 26, 2016
Priority date	Nov 29, 2006
Publication date	Jun 26, 2018
Grant date	Jun 26, 2018

Abstract

The present invention is directed to nucleic acid molecules containing a loop sequence designed to circumvent exportin-5 mediated export, and methods using these novel molecules.

First claim

What is claimed is:

1. An isolated nucleic acid molecule comprising
a first portion, wherein the first portion is no more than 30 nucleotides in length;
a second portion, wherein the second portion has a sequence that is complementary to the first portion; and
a loop portion comprising a sequence designed to circumvent exportin-5 mediated export, wherein the loop portion comprises between 12 and 32 nucleotides of SEQ ID NO:1;
wherein the first portion and the second portion are operably linked by means of the loop portion to form a hairpin structure comprising a duplex structure and a loop structure.

2. The nucleic acid molecule of claim 1 , wherein the loop portion is a 31 or 32 nucleotide L1 motif.

3. The nucleic acid molecule of claim 1 , wherein the loop portion consists of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

4. The nucleic acid molecule of claim 1 , wherein the loop portion comprises between 12 and 32 contiguous nucleotides of SEQ ID NO:1.

5. The nucleic acid molecule of claim 1 , wherein the loop portion consists of SEQ ID NO:5.

6. The nucleic acid molecule claim 1 , wherein the duplex is less than 30 nucleotides in length.

7. The nucleic acid molecule claim 1 , wherein the duplex contains from 19 to 25 nucleotides.

8. The nucleic acid molecule claim 1 , wherein the molecule is a short hairpin RNA (shRNA).

9. The nucleic acid molecule claim 1 , wherein the molecule is a microRNA (miRNA).

10. An expression cassette comprising a sequence encoding a nucleic acid molecule comprising:
a promoter;
a first portion, wherein the first portion is no more than 30 nucleotides in length;
a second portion, wherein the second portion has a sequence that is complementary to the first portion; and
a loop portion comprising a sequence designed to circumvent exportin-5 mediated export wherein the loop portion comprises between 12 and 32 nucleotides of SEQ ID NO:1;
wherein the first portion and the second portion are operably linked by means of the loop portion to form a hairpin structure comprising a duplex structure and a loop structure.

11. A vector comprising the expression cassette of claim 10 .

12. A cell comprising the nucleic acid of claim 1 .

13. A cell comprising the expression cassette of claim 10 .

14. A cell comprising the vector of claim 11 .

15. A method of reducing the expression of a gene product in a cell, comprising contacting a cell with the nucleic acid of claim 1 .

16. A method of reducing the expression of a gene product in a cell, comprising contacting a cell with the expression cassette of claim 10 .

17. A method of reducing the expression of a gene product in a cell, comprising contacting a cell with the vector of claim 11 .

18. The vector of claim 11 , wherein the vector is a viral vector.

19. The vector of claim 11 , wherein the viral vector is an AAV vector.

Assignees

• Univ Iowa Res Found

Inventors

• Harper Scott
• Davidson Beverly L

Classifications

• C12N2310/111

  spanning the whole gene, or a large part of it · CPC title

• C12N2310/531

  Stem-loop; Hairpin · CPC title

• C12N15/111 Primary

  General methods applicable to biologically active non-coding nucleic acids · CPC title

• C12N2310/53

  partially self-complementary or closed · CPC title

• C12N2320/50

  Methods for regulating/modulating their activity · CPC title